The Hemophilus influenzae Hap Autotransporter Is a Chymotrypsin Clan Serine Protease and Undergoes Autoproteolysis via an Intermolecular Mechanism

Doran L. Fink, Leslie D. Cope, Eric J. Hansen, Joseph W. St. Geme

Research output: Contribution to journalArticle

52 Scopus citations

Abstract

The Hemophilus influenzae Hap adhesin is an autotransporter protein that undergoes an autoproteolytic cleavage event resulting in extracellular release of the adhesin domain (Haps) from the membrane-associated translocator domain (Hapβ). Hap autoproteolysis is mediated by Ser243 and occurs at LN1036-7 and to a lesser extent at more COOH-terminal alternate sites. In the present study, we sought to further define the mechanism of Hap autoproteolysis. Site-directed mutagenesis of residues His98 and Asp140 identified a catalytic triad conserved among a subfamily of autotransporters and reminiscent of the SA (chymotrypsin) clan of serine proteases. Amino-terminal amino acid sequencing of histidine-tagged Hapβ species and site-directed mutagenesis established that autoproteolysis occurs at LT1046-7, FA1077-8, and FS1067-8, revealing a consensus target sequence for cleavage that consists of ((Q/R)(A/S)X(L/F)) at the P4 through P1 positions. Examination of a recombinant strain co-expressing a Hap derivative lacking all cleavage sites (HapΔ1036-99) and a Hap derivative lacking proteolytic activity (HapS243A) demonstrated that autoproteolysis occurs by an intermolecular mechanism. Kinetic analysis of Hap autoproteolysis in bacteria expressing Hap under control of an inducible promoter demonstrated that autoproteolysis increases as the density of Hap precursor in the outer membrane increases, confirming intermolecular cleavage and suggesting a novel mechanism for regulation of bacterial adherence and microcolony formation.

Original languageEnglish (US)
Pages (from-to)39492-39500
Number of pages9
JournalJournal of Biological Chemistry
Volume276
Issue number42
DOIs
StatePublished - Oct 19 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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